Background: Pulmonary hypertension is a devastating vascular disease for which no current cure exists. Despite a number of FDA-approved therapies, survival remains low and no current therapy reverses the vascular remodeling in the pulmonary vasculature responsible for disease progression. The pulmonary vascular endothelium is thought to play an important role in initiating and promoting the PH-associated vascular remodeling. However, how this occurs remains elusive and the phenotypic signature of the lung endothelium as the disease progresses remains a challenging process to understand. We have recently shown that changes in reactive oxygen species (ROS) driven by NADPH oxidase 1 (Nox1) are involved in promoting pulmonary endothelial proliferative mechanisms in PH. We recently also linked Nox1 activity to the scaffolding protein ERM-binding phosphoprotein 50 (EBP50, aka NHERF1) in systemic vascular cells, suggesting a role for EBP50 in the pulmonary vasculature. Preliminary data demonstrate that in vitro modulation of human pulmonary arterial endothelial cells with PH- related stimuli affects EBP50 expression and that in vitro and in vivo interruption in EBP50 leads to exacerbated PH-related responses. Analysis utilizing PCR pathway arrays revealed upregulation of endothelial-to-mesenchymal transition (EndMT) transcription factors potentially in an EBP50 dependent manner in a PH mouse model. This proposal will test the causality of this link and will assess the contribution of EndMT to vascular remodeling in PH and test whether it occurs in an EBP50-dependent mechanism. Hypothesis: PH-induced perturbation of EBP50 homeostasis promotes phenotypic reprogramming of pulmonary endothelial cells leading to increased EndMT and vascular remodeling. Specific Aims: 1-) To test whether attenuation of EBP50 is causally linked to development or worsening of PH; 2-) To test whether EBP50 is an upstream negative modulator of EndMT in the pulmonary vascular endothelium; and 3) To test whether therapeutic rescue of EBP50 homeostasis in attenuates EndMT and in vivo manifestations of PH in preclinical models. Gain- and loss- of function techniques will be used to sequentially and combinatorially target EBP50 and EndMT-related transcription factor in vitro in pulmonary vascular cells subjected to PH-related stimuli. Knockout animals and administration of recombinant proteins and/or blocking antibodies will be used in in vivo models of PH. Finally, lineage tracing experiments will be used to assess EndMT in PH models. Significance: Current PH drugs neither prevent nor reverse disease progression. If successful, the proposal will uncover novel molecular mechanisms and reprogramming pathways in the pulmonary vasculature that can be targeted for future PH drug development.